Dietary Addition of Cellular Metabolic Intermediates ...

Dietary Addition of Cellular Metabolic Intermediates and Carcass Fat Deposition in Broilers P. LESSARD,1 M. R. LEFRANCOIS,1-2 and J. F. BERNIER1'3 Dfyartement de Zootechnie, University Laval, Ste-Foy, Quebec, Canada, G1K 7P4 and

ABSTRACT Ninety-six 1-day-old male broilers were fed a diet containing 0, 2, 4, 6, 8, or 10% of a 1:1 mixture of pyruvic acid (PY) and dihydroxyacetone (DH) for ad libitum consumption for 42 days. Feed intake, body weight gain, and feed efficiency decreased linearly (P < .001) with increasing levels of PY and DH. There were no significant differences among treatments for abdominal fat percentage. Carcass chemical analysis revealed small but significant (P < .05) differences among dietary treatments for protein and fat percentages. In a second experiment, 1921-day-old male broilers were fed diets containing 5% of PY, lactic acid (LA), citric acid (CI), DH, or glycerol (GY) or mixtures (1:1) of DH or GY in combination with each organic acid. Bird performance was impaired (P < .05) by PY or CI but not by DH or GY. Lactic acid reduced (P < .05) feed intake by 9% without affecting weight gain. Lactic acid plus DH, CI plus DH, and CI plus GY mixtures decreased (P < .05) bird performance but other combinations had no effect. Pyruvic acid or CI decreased abdominal fat and carcass lipid percentages. Dihydroxyacetone increased (P < .05) carcass lipid percentage and GY increased (P < .05) abdominal fat percentage. Lactic acid plus DH increased (P < .05) carcass lipid percentage. Only PY and CI decreased carcass fat deposition, but they also impaired broiler performance. {Key words: broilers, carcass composition, organic acids, dihydroxyacetone, glycerol) 1993 Poultry Science 72:535-545

pyruvate and dihydroxyacetone, two hydrogen acceptors, prevented the inSome organic metabolites have been crease in the cytoplasmic dihydronicotinaused as dietary supplements to reduce de mide adenine dinucleotide (NADH) over novo fatty acid synthesis and subsequent nicotinamide adenine dinucleotide fat deposition in animals. A dietary 1:1 (NAD+) ratio normally observed with mixture of pyruvate and dihydroxyace- ethanol oxidation (Lieber, 1973). However, tone prevents fatty liver caused by chronic this hypothesis was rejected when a lacalcohol ingestion in rats (Stanko et al, 1978; Goheen et al, 1981; Rao et al, 1984). tate and glycerol mixture, the reduced Stanko et al (1978) hypothesized that forms of pyruvate and dihydroxyacetone, respectively, inhibited liver fat accumulation in rats fed alcohol (Rao et al, 1984). Furthermore, Stanko et al. (1978) reported that pyruvate or dihydroxyacetone fed Received for publication August 11, 1992. Accepted for publication December 8, 1992. alone did not inhibit liver fat accumulaiUniversite Laval. tion, whereas Rao et al. (1984) observed 2 Agriculture Canada. 3 To whom correspondence should be addressed. that pyruvate was effective in reducing fat INTRODUCTION

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Centre for Food and Animal Research, Agriculture Canada, Ottawa, Ontario, Canada, K1A 0C6

536

LESSARD ET AL.

To the authors' knowledge, the effects of pyruvate, dihydroxyacetone, and related compounds on fat deposition in broiler chickens have not been reported. The objectives of the present study were to determine: 1) the optimal level of pyruvic acid and dihydroxyacetone required to reduce fat deposition in broilers without any reduction in growth rate and protein deposition; and 2) the effect of pyruvic acid, lactic acid, citric acid, dihydroxyacetone, and glycerol, fed individually or in combination, on growth performance and carcass fat deposition in broilers. MATERIALS AND METHODS Experiment 1

Ninety-six 1-day-old Arbor Acres x Arbor Acres male broiler chicks (vaccinated against Marek's disease) were obtained from a commercial hatchery.4 Chicks were wing-banded and assigned randomly to one of six treatments with eight replicates of two chicks per treatment in a completely randomized block design with initial weight as the blocking factor. They were housed in pairs in wire mesh cages. Room temperature was maintained at 32 C for 7 days and gradually reduced to 20 C at 6 wk. Broilers were exposed to a lightdark cycle of 23 h light (L):l h dark (D) for the first 7 days, then to a 12L:12D cycle until 6 wk. Broilers were fed a starter diet from Day 1 to 21, and a finisher diet to 42 days. Diets were formulated according to NRC (1984) recommendations. The experimental diets contained 90% of a basal diet (Table 1) and either 0,2,4, 6, 8, or 10% of a 1:1 mixture of pyruvic acid5 and dihydroxyacetone5 completed to 100% with cornstarch. Individual body weights and feed intake of each pair were recorded weekly. Feed and water were supplied for ad libitum consumption. At the end of the trial, birds were weighed, held for 12 h without feed or water, then slaughtered, after which feathers, feet, heads, and necks were removed. Carcasses were placed in polyethylene bags and cooled overnight at 4 C. The following day, the liver and abdominal fat pad (around the cloaca, the intestines and 4 Couvoir Scott Ltee, Scott Jonction, PQ, Canada, the gizzard) were removed, weighed, and GOS 3G0. 5 Omega Chemical Co., Ste-Therese, PQ, Canada, frozen at -20 C. The remaining carcass was eviscerated, weighed, and frozen at -20 C GIN 1S7.


deposition but that dihydroxyacetone was not. Glycerol prevented fatty liver in rats fed alcohol and lactate reduced partially hepatic triacylglycerol concentration (Rao et ah, 1984). The effects of pyruvate and dihydroxyacetone on body fat deposition have also been studied in growing animals fed diets without alcohol. Replacement of dextrin with pyruvate and dihydroxyacetone in a liquid diet reduced body fat by 32% without affecting protein deposition in growing rats (Stanko and Adibi, 1986). Rats consuming pyruvate and dihydroxyacetone had a lower rate of lipid synthesis in adipose tissue and increased heat production at the expense of storage of lipids. Replacement of 4% cornstarch by a 1:3 mixture of pyruvate plus dihydroxyacetone reduced average backfat thickness by 12% and muscle fat content by 6% in finishing swine without reducing growth rate or protein deposition (Stanko et ah, 1989). Lipid synthesis has also been studied in growing chicks and rats fed glycerol. Chickens fed a diet containing 22.5% glycerol for 3 wk showed a significant reduction in hepatic lipogenesis (Lin et ah, 1976). In rats, a diet containing 22.5% glycerol reduced lipogenesis in one experiment but had no effect in the other (Lin et ah, 1976). Reduced lipogenesis was associated with lower hepatic fatty acid synthetase and malic enzyme activities in chickens but with higher activities in rats (Lin et ah, 1976). The effect of lactic acid on fat deposition has only been studied in rats fed alcohol. Jacob and Blair (1990) reported that calcium lactate reduced broiler feed intake when fed at 5 or 7.5% of the diet, but they did not report data on body composition of these birds. The influence of dietary addition of citric acid on lipid synthesis has not been reported in chickens. The cellular concentration of citric acid is important in the allosteric control of de novo lipid synthesis (Goodridge, 1973; Donaldson, 1979; Mayes, 1989).

METABOLIC INTERMEDIATES AND FAT DEPOSITION IN BROILERS TABLE 1. Basal diet composition1 Ingredients and composition

529.2 258.6 84.1 50.0 40.0 15.3 13.9 3.3 2.8 1.0 .7 .8 .4 25.6 3,190

Finisher (g/kg) 643.1 168.1 85.2 50.0 20.0 12.9 12.6 3.3 2.8 1.2 .8 22.2 3,190

ground twice with a meat grinder6 using a 5-mm die. Ground tissues were mixed for 1 min at low speed in a small mixer,6 and two samples of approximately 250 g were weighed precisely, autoclaved7 in covered aluminum trays for 2 h at 121 C, then transferred quantitatively and homogenized for 1 min in a blender8 with water added to produce a free-flowing homogenate. Samples were then freezedried9^ for 3 days. Dry samples were allowed to equilibrate with room humidity for 24 h before weighing and were ground with a domestic food processor10 prior to chemical analysis. Livers were freezedried9 for 2 days, left to adjust to room humidity for 24 h, then weighed and ground in a coffee grinder11 prior to chemical analysis.

iRepresenting 90% of experimental diets. Provided the following nutrients per kilogram of Experiment 2 diet: vitamin A, 10,000 IU; cholecalciferol, 2,500 IU; One hundred and ninety-two 1-day-old vitamin E, 35 IU; menadione, 2 mg; pantothenic acid, 12 mg; riboflavin, 6 mg; folic acid, .8 mg; niacin, 65 mg; Ross x Peterson male broiler chicks (vaccithiamin, 3 mg; pyridoxine, 4 mg; vitamin Bj2/ -018 mg; nated against Marek's disease) were obbiotin, .15 mg; iodine, .5 mg; manganese, 70 mg; zinc, tained from a commercial hatchery.4 Chicks 80 mg; iron, 100 mg; copper, 10 mg; selenium, .3 mg. were wing-banded and assigned randomly 3 Hoffmann La Roche Ltd. (Mississauga, ON, to 1 of 12 treatments with eight replicates of Canada, L5N 6L7); 150 g/kg of lasalocid sodium. two chicks per treatment in a completely 2

until further dissection. Kidneys remained attached to the carcass. Carcasses were thawed for 16 h at 4 C and dissected. Legs and deboned breast muscle were weighed individually after removing skin and adhering fat, then all pieces were frozen together at -20 C. Frozen carcasses were cut into small pieces and

6 Hobart Manufacturing Co. Ltd., Don Mills, ON, Canada, M3A 1B1. 7 American Sterilizer Canada Inc., Brampton, ON, Canada, L6W 2B9. 8 Waring Products Co., New Hartford, CT 06057. 9 Model 10MR-TR-LP, Virtis Co., Gardiner, NY 12525. 10 Model FM-100, Sunbeam Corp. Ltd., Toronto, ON, Canada, M5M 3Z1. "Chemical Rubber Co., Cleveland, OH 44128. 12 Model 524-A, Precision Scientific, Chicago, IL 60647. "Model F-A1730, Thermolyne Co., Dubuque, IA 52001. 14 N. Foss Electric, Hillered, Denmark. 15 Soxtec System HT1043 extraction unit, Tecator AB, Hoganas, Sweden.

randomized block design with initial weight as the blocking factor. Birds were housed in pairs and handled as described in Experiment 1. Broilers were fed the same basal diets as in Experiment 1 (Table 1) from Day 1 to 42 to be consumed ad libitum. The 12 experimental diets were 90% basal diet (Table 1), 5% cornstarch, and 5% additives as described in Table 2. All other manipulations were as described in Experiment 1, except that there was no feed withdrawal period before slaughter and the legs and breast muscle were not weighed separately. Chemical Analysis

To complete the determination of dry matter and ash content of the carcass and liver, freeze-dried samples were dried overnight at 70 C in a vacuum oven,12 weighed, then incinerated overnight at 550 C in a muffle furnace,13 and weighed again. Nitrogen content of the dried carcass and liver were determined by the Kieldahl method using a Kjel-Foss apparatus.14 Fat content of the dried carcass and liver samples was determined by15extraction with anhydrous diethyl ether.


Com Soybean meal Corn gluten meal Fish meal, menhaden Animal fat Dicalcium phosphate Calcium carbonate Salt Vitamin-mineral premix2 DL-methionine L-lysine-HCl Avatec 15%3 Choline chloride Calculated composition Protein, % ME, kcal/kg

Starter

537

LESSARD ET AL.

Statistical Analysis

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Feed intake from Day 1 to 42 decreased linearly (P < .001) with increasing levels of pyruvic acid and dihydroxyacetone (Table 3). There was an 11% difference in feed intake between the control and the lowest feed intake (8% incorporation of metabolites). In previous studies with rats and pigs, the effect of pyruvic acid and dihydroxyacetone on feed intake could not be determined because treated and control animals were pair-fed (Stanko et ah, 1978, 1989; Goheen et ah, 1981; Rao et ah, 1984; Stanko and Adibi, 1986). Langhans et al. (1985a) reported that subcutaneous injections of pyruvate, lactate, glycerol, and malate reduced feed intake in rats, whereas dihydroxyacetone and oxaloacetate injections did not. They hypothesized that the generation of reducing equivalents by the first oxidative step in the utilization of pyruvate, lactate, glycerol, and malate reduces feed intake. However, the first oxidation step of dihydroxyacetone and oxaloacetate does not produce reducing equivalents. Selective hepatic vagotomy indicated that the hypophagic effect of pyruvate, lactate, glycerol, and malate in rats is mediated through hepatic neural structures (Langhans et ah, 1985b). The reduced feed intake related to increasing levels of trioses could also be attributed to diet acidity caused by the addition of pyruvic acid. Gentle (1978) reported that the chicken is able to discriminate between the taste of water and of


Growth performance and carcass and liver composition data were analyzed with the General Linear Models procedure of SAS® software (SAS Institute, 1989) according to a completely randomized block design. In Experiment 1, linear, quadratic, and cubic effects associated with increasing levels of the pyruvic acid and dihydroxyacetone mixture were tested with orthogonal contrasts (Steel and Torrie, 1980). In Experiment 2, significant differences among treatment means were analyzed using Waller Duncan's multiple range test (Steel and Torrie, 1980).

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539

METABOLIC INTERMEDIATES AND FAT DEPOSITION IN BROILERS

TABLE 3. Performance and carcass characteristics of broilers fed diets containing graded levels of a 1:1 mixture of pyruvic acid and dihydroxyacetone from 1 to 42 days of age, Experiment 1 Level of pyruvic acid and dihydroxyacetone Variable

0%

2% 82

3,714 2,094 .564

6%

10%

8%

7

8

8

8

7

3,763 1,997 .530

3,624 1,982 .547

3,600 1,898 .527

3,323 1,746 .526

3,375 1,810 .536

1,432 1,361 1,328 1,258 1,150 1,196 3.8 4.0 3.8 3.7 3.5 3.9 2.7 2.8 3.0 3.0 3.0 3.0 29.8 29.6 29.8 31.1 30.1 30.1 19.4 19.3 19.0 17.2 17.5 18.1

X

SD

L

176 103 .017

*** NS *** NS *** **

71

Q

C

NS NS NS

NS NS NS NS NS NS NS NS NS NS NS ** NS NS

***

.5 .3 1.4 1.3

quadratic effect; C = cubic effect.

L = linear effect; Q

2n.

**P < .01. ***P < .001.

diluted acetic acid. Chickens refuse to drink 5 N acetic acid solutions (Gentle, 1978). Pinchasov and Jensen (1989) reported that 3% propionic acid in the diet of chicks reduced feed intake significantly. However, 3% calcium propionate or 3% propionic acid neutralized with KOH also reduced feed intake significantly (Pinchasov and Jensen, 1989).

Body weight gain decreased linearly (P < .001) with increasing levels of pyruvic acid plus dihydroxyacetone (Table 3). There were also linear and quadratic mixture concentration effects (P < .01) on feed efficiency. Stanko a n d Adibi (1986) reported a decrease in body weight gain of rats fed a diet containing pyruvate and dihydroxyacetone. They attributed this

TABLE 4. Carcass and liver chemical analysis of broilers fed diets containing graded levels of a 1:1 mixture of pyruvic acid and dihydroxyacetone from 1 to 42 days of age, Experiment 1 Probability 1

Level of pyruvic acid and dihydroxyacetone Variable Carcass analysis, % DM Crude protein 3 Ether extract 3 Ash 3 Liver analysis, % DM Crude protein 3 Ether extract 3 Ash 3 J

3

DM basis. *P < .05. **P < .01.

L

Q

C

36.2 46.9 40.4 8.4

.9 1.8 2.4 .4

NS NS NS

NS NS NS NS

if

* **

26.2 74.3 15.5 5.1

1.5 4.8 4.1 .3

NS NS NS NS

NS NS NS NS

NS NS NS NS

2%

4%

6%

8%

10%

82

7

8

8

8

7

36.2 48.9 38.9 8.0

37.0 47.2 41.9 7.9

36.5 47.9 39.9 8.3

36.8 45.5 42.0 8.1

35.3 49.6 37.0 8.8

25.7 77.0 12.7 5.1

26.7 76.8 13.8 5.1

26.5 72.4 16.7 4.9

26.1 75.2 15.3 5.1

26.0 72.9 14.1 5.1

L = linear effect; Q = quadratic effect; C = cubic effect.

2n.

SD

0%

XX-

NS


Performance Feed intake, g Weight gain, g Feed efficiency, g:g Carcass characteristics Carcass weight, CW, g Abdominal fat, %CW Liver weight, %CW Leg weight, %CW Breast weight, %CW

4%

Probability1

540

LESSARD LT AL. TABLE 5. Performance of broilers fed diets containing 5% of different metabolites from 1 to 42 days of age, Experiment 2

n

Control Pyruvic acid (PY) Lactic acid (LA) Citric acid (CI) Dihydroxyacetone (DH) Glycerol (GY) PY + DH LA + DH CI + DH PY + GY LA + GY CI + GY SD

8 8 7 8 8 8 8 8 8 8 8 8

Feed intake

Weight gain

Feed efficiency

2,038*' 1,656* l,990bcd 1,232' l,948bcd 2,074* l,956bcd l,635e l,872d l,971bcd 2,160a l,883cd 171

(g:g) ,570bc .583* .608* .560bc .566bc .580b .570bc .548c .587* .570bc .585* .573bc .025

(g) 3,580* 2,837' 3,265 .05) to those fed the controls (Tables 6 and 7). The lack of effect of these combinations on body composition may be explained by the opposite effects of organic acids and dihydroxyacetone or glycerol on fat deposition. It could also be attributed to the lower level of incorporation of organic acids and dihydroxyacetone or glycerol in the combinations.

METABOLIC INTERMEDIATES AND FAT DEPOSITION IN BROILERS

mucosa and liver of the chicken (Gallus domesticus). Int. J. Biochem. 3:322-328. Rao, G. A., D. E. Riley, and E. C. Larkin, 1984. Fatty liver caused by chronic alcohol ingestion is prevented by dietary supplementation with pyruvate or glycerol. Lipids 19:583-588. SAS Institute, 1989. SAS/STAT® User's Guide, Version 6, 4th ed. Vol. 2. SAS Institute Inc., Cary, NC. Stanko, R. T., and S. A. Adibi, 1986. Inhibition of lipid accumulation and enhancement of energy expenditure by the addition of pyruvate and dihydroxyacetone to a rat diet. Metabolism 35: 182-186. Stanko, R. T., T. L. Ferguson, C. W. Newman, and R. K. Newman, 1989. Reduction of carcass fat in swine with dietary addition of dihydroxyacetone and pyruvate. J. Anim. Sci. 67:1272-1278. Stanko, R. T., H. Mendelow, H. Shinozuka, and S. A. Adibi, 1978. Prevention of alcohol-induced fatty liver by natural metabolites and riboflavin. J. Lab. Clin. Med. 91:228-235. Steel, R.G.D., and J. H. Torrie, 1980. Principles and Procedures of Statistics. A Biometrical Approach, 2nd ed. McGraw-Hill Publishing Co., New York, NY. Tzeng, R., and W. A. Becker, 1981. Growth patterns of body and abdominal fat weights in male broiler chickens. Poultry Sci. 60:1101-1106.


Auton. Nerv. Syst. 13:255-262. Leeson, S., and J. D. Summers, 1980. Production and carcass characteristics of the broiler chicken. Poultry Sci. 59:786-798. Leveille, G. A., D. R. Romsos, Y. Y. Yeh, and E. K. O'Hea, 1975. Lipid biosynthesis in the chick. A consideration of site of synthesis, influence of diet and possible regulatory mechanisms. Poultry Sci. 54:1075-1093. Lieber, C. S., 1973. Hepatic and metabolic effects of alcohol. Gastroenterology 65:821-846. Lin, M. H., D. R. Romsos, and G. A. Leveille, 1976. Effect of glycerol on lipogenic enzyme activities and on fatty acid synthesis in the rat and chicken. J. Nutr. 106:1668-1677. Mayes, P. A., 1989. Regulation du metabolisme des glucides. Pages 209-221 in: Precis de Biochimie de Harper, 7th ed. D. K. Granner, P. A. Mayes, R. K. Murray, V. W. Rodwell, ed. Les Presses de l'Universite Laval et Eska, Quebec-Paris, PQ, Canada. National Research Council, 1984. Nutrient Requirements of Poultry. 8th rev. ed. National Academy Press, Washington, DC. Pinchasov, Y., and L. S. Jensen, 1989. Effect of shortchain fatty acids on voluntary feed intake of broiler chicks. Poultry Sci. 68:1612-1618. Pritchard, P. J., and D.J.W. Lee, 1972. The effect of dietary citrate on glycolysis in the intestinal

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